Glucagon-like peptide 2 (GLP-2) is a 33 amino acid residue peptide produced in intestinal L-cells and released following nutrient intake. The amino acid sequence of the human GLP-2 peptide is given in FIG. 1.
The GLP-2 peptide is a product of the proglucagon gene. Proglucagon is expressed mainly in the pancreas and the intestine and to some extent in specific neurons located in the brain. The posttranslational processing of proglucagon is however different in pancreas and intestine (FIG. 2). In the pancreas proglucagon is processed mainly to Glucagon Related Pancreatic Polypeptide (GRPP), Glucagon and Major Proglucagon Fragment. In contrast to this the processing in the intestine results in Glicentin, Glucagon-Like Peptide 1 (GLP-1) and Glucagon-Like Peptide 2 (GLP-2).
The GLP-2 peptide is rather well conserved between species although sequence differences exists (FIG. 3). The pig GLP-2 peptide for example has 4 substitutions as compared to the human GLP-2. Interestingly the mouse GLP-2 has only two substitutions compared to the human GLP-2.
GLP-2 is secreted from the L-cells in the small and large intestine. This secretion is regulated by nutrient intake. The plasma concentration of GLP-2 in normal fasting subjects is around 15 pM increasing to around 60 pM after a mixed meal.
The actions of GLP-2 are transduced by a recently cloned glucagon-like peptide-2 receptor. The GLP-2 receptor represents a new member of the G protein-coupled 7TM receptor superfamily. The GLP-2R is expressed in a highly tissue-specific manner predominantly in the gastrointestinal tract (FIG. 4) and GLP-2R activation is coupled to increased adenylate cyclase activity. Cells expressing the GLP-2R responds to GLP-2, but not to other peptide of the glucagon family (Glucagon, GLP-1 and GIP).
In the rat the GLP-2R has also been reported to be expressed in the brain or more specific the dorsomedial hypothalamic nucleus. This part of the brain is normally thought to be involved in feeding behaviour and it has been shown that GLP-2 inhibits food intake when injected directly into the brain.
Induction of intestinal epithelial proliferation by GLP-2 was demonstrated (Drucker, D. J. et al (1996) Proc. Natl. Acad. Sci. USA 93: 7911-7916) and treatment of gastrointestinal diseases by cells grown in medium containing GLP-2 was disclosed (Drucker, D. J and Keneford, J. R., WO 96/32414).
WO 97/31943 relates to GLP-2 peptide analogs and the use of certain GLP-2 peptide analogs for appetite suppression or sateity induction.
WO 98/08872 Relates to GLP-2 derivatives comprising a lipophilic substituent.
WO 96/32414 and WO 97/39031 relates to specific GLP-2 peptide analogs.
WO 98/03547 relates to specific GLP-2 peptide analogs, which exhibit antagonist activity
While much attention has been focused on the pharmacological properties of GLP-2 compounds, hitherto little is known about their physico-chemical and solution structural properties. Such knowledge is a prerequisite for rational handling during e.g. production, purification and formulation work and is eventually important for understanding of the structural basis for the protraction mechanism.
It is an important technical challenge to ensure prolonged stability during storage (shelf life) of many protein based drug products due to the inherent lability of macromolecules. Hence, proteins are sensitive to both chemical and physical degradation unlike many small molecules. Chemical degradation involves covalent bonds, such as hydrolysis, racemization, oxidation or crosslinking. Physical degradation involves conformational changes relative to the native structure, which includes loss of higher order structure, aggregation, precipitation or adsorption to surfaces. GLP-2 is known to be prone to instability due to aggregation. Both degradation pathways may ultimately lead to loss of biological activity of the protein drug.
GLP-2 and analogs of GLP-2 and fragments thereof are potentially useful i.a. in the treatment of gastrointestinal diseases. However, solubility limitations and the low stability against the actions of endogenous diaminopeptidyl peptidase limits the usefulness of these compounds, and thus there still is a need for improvements in this field.
In WO 99/43361 are disclosed certain pharmaceutical formulations comprising GLP-2 having a lipophilic substituent.
In WO 01/49314 are disclosed a formulations comprising GLP-2 or GLP-2 analogs in a physiological buffer containing L-histidine and a bulk agent selected from a group consisting of mannitol and sucrose, at pH 5.5 to 7.9.
GLP-2 peptides and derivatives thereof are useful in the treatment of gastrointestinal disorders. However, the high clearance limits the usefulness of these compounds, and thus there still is a need for improvements in this field. Accordingly, it is an object of the present invention to provide peptides of GLP-2 and derivatives thereof which have a protracted profile of action relative to native GLP-2, while still retaining the GLP-2 activity. It is a further object of the invention to provide a pharmaceutical composition comprising a compound according to the invention and to use a compound of the invention to provide such a composition. Also, it is an object of the present invention to provide a method of treating gastrointestinal disorders.